Currently, there is a need for novel, potent, and selective agents to prevent detrimental effects upon cells due to DNA damage, such as caused by chemotherapy, radiation, ischemic event, including ischemia-reperfusion injury and organ transplantation, and the like. There is also a need for pharmacological tools for the further study of the physiological processes associated with intracellular DNA damage.
p53, the product of the p53 tumor suppressor gene, is a multifunctional tumor suppressor protein, involved in the negative control of cell growth. In response to a variety of stressors, p53 induces growth arrest or apoptosis, thereby eliminating damaged and potentially dangerous cells. T. M. Gottleib et al., Biochim. Biophys, Acta, 1287, 77 (1996). Mutations in the p53 gene are frequently associated with the metastatic stage of tumor progression, and lack of functional p53 is accompanied by rapid tumor progression, resistance to anti-cancer therapy and increased tumor angiogenesis. See, e.g., A. J. Levine et al., Br. J. Cancer, 69, 409 (1994); R. J. Steele et al., Br. J. Surg., 85, 1460 (1998); C. Cordon-Cardo et al., Surg. Oncol., 13, 319 (1997). p53 deficiency in mice is associated with a high frequency of spontaneous cancers. L. A. Donehower et al., Nature, 356, 215 (1992); T. Jacks et al., Curr, Biol., 4, 1 (1994). On the basis of these reports, the inactivation of p53 was viewed as an unfavorable event, and it has been speculated that cancer can be inhibited by restoration of p53 function.
However, in mice, the level of expression of p53 has been reported to be directly related to the susceptibility of normal tissues such as hematopoietic cells, intestinal epithelia and the testis, to damage by anti-cancer therapy. A. Rogel et al., Mol. Cell, Biol., 5, 2851 (1985); P. Schmidt et al., Development, 113, 857 (1991), D. Schwartz et al., Oncogene, 8, 1487 (1993). p53-dependent apoptosis occurs in such sensitive tissues shortly after gamma-irradiation, and p53-deficient mice survive higher doses of irradiation than do wild-type animals. E. Komarov et al., EMBO J., 16, 1391 (1997); J. H. Hendry et al., J. Radiat, Biol., 70, 677 (1996); V. A. Tron et al., Am. J. Pathol, 153, 579 (1998). These data indicate that p53 is a determinant of the toxic side effects of anti-cancer therapy, and thus may be an appropriate target for suppression, which in turn, may reduce the damage to normal tissues. See, E. A. Komarov et al., Semin. Cancer Biol., 8, 389 (1998).
Recently, P. G. Komarov et al., Science, 285, 1733 (1999), reported that a compound of formula: 
which they named PFTxcex1, protected mice from lethal genotoxic stress associated with 6-8 Gy of gamma radiation, without itself promoting the formation of tumors or affecting the sensitivity of the tumors to radiation. These authors suggested that PFTxcex1 might be useful to reduce the side effects of radiation therapy or chemotherapy for human cancers. PFTxcex1 is a known 2-imino-tetrahydro-benzothiazole that was first prepared by A. Singh et al., Ind. J. Chem., 1413, 997 (1976) as a potential antihelminthic agent. However, a continuing need exists for compounds that can protect mammalian cells from the damaging effects of chemotherapy and irradiation, or in other situations in which it is desirable to protect tissue from the consequences of clinical or environmental stress.
The present invention provides compounds that act to suppress p53 activity in mammalian cells, and a method to effectively suppress p53 activity in the cells of a mammal subject to a stress or pathology that is ameliorated by such suppression. Accordingly, there is provided a method of p53 suppression comprising administering to a mammal in need of said suppression an effective amount of a compound of formula (I): 
wherein
R1, R2 and R3 are independently hydrogen, halo, hydroxy, cyano, N(Ra)(Rb), S(Ra), NO2, (C1-C6)alkyl, (C1-C6)alkoxy, (C2-C6)alkynyl, (C2-C6)alkenyl, (C2-C7)alkanoyl, (C2-C7)alkanoyloxy, or (C3-C7)cycloalkyl or R1 and R2 taken together are benzo, optionally substituted by R1, (C3-C5)alkylene or methylenedioxy; wherein Ra and Rb are each independently hydrogen, (C1-C3)alkyl, (C2-C4)alkanoyl, phenyl, benzyl, or phenethyl; or Ra and Rb together with the nitrogen to which they are attached are a 5-6 membered heterocyclic ring, preferably a pyrrolidino, piperidino or morpholino ring;
Ar is aryl or heteroaryl, optionally substituted with 1-5, preferably 1-2, halo, CF3, hydroxy, CN, N(Ra)(Rb), (C1-C6)alkyl, (C1-C6)alkoxy, (C2-C7)alkanoyl, (C2-C7)alkanoyloxy, (C3-C7)cycloalkyl, (C2-C6)alkanoyl, (C2-C6)alkenyl, or phenyl.
Y is oxy (xe2x80x94Oxe2x80x94), S(O)0-2, C(R1)(R3), N(Ra), or xe2x80x94Pxe2x80x94;
or a pharmaceutically acceptable salt thereof.
The invention also provides novel p53 suppressor compounds, as well as pharmaceutical compositions comprising novel compounds of formula I, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable diluent or carrier. Such compounds can be represented by compounds of formula (I), with the proviso that when Y is S, Ar is not phenyl (C6H5).
Additionally, the invention provides a therapeutic method for preventing or treating a pathological condition or symptom in a mammal, such as a human, wherein the activity of p53 is implicated and antagonism or suppression of its action is desired, comprising administering to a mammal in need of such therapy, an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof. Such pathological conditions or symptoms include blocking, moderating or reversing the deleterious effects of chemotherapeutic agents, particularly those which damage DNA; radiation, particularly radiation therapy (gamma-, beta- or UV-radiation), ischemic event, including stroke, infarct, ischemia-reperfusion injury and ischemia due to organ, tissue or cell transplantation; environmental pollution or contamination and the like.
The invention provides a compound of formula (I) for use in medical therapy as well as the use of a compound of formula (I) for the manufacture of a medicament for the treatment of a pathological condition or symptom in a mammal, such as a human, which is associated with p53-induced cellular damage, i.e., with unwanted apoptosis.
The invention also includes a method for binding a compound of formula (I) to cells and biomolecules comprising p53 receptors, in vivo or in vitro, comprising contacting said cells or biomolecules with an amount of a compound of formula (I) effective to bind to said receptors. Cells or biomolecules comprising ligand-bound p53 receptor sites can be used to measure the selectivity of test compounds for specific receptor subtypes, or can be used as a tool to identify potential therapeutic agents for the treatment of diseases or conditions associated with p53 activation, by contacting said agents with said ligand-receptor complexes, and measuring the extent of displacement of the ligand and/or binding of the agent, by methods known to the art.
As used herein, the term xe2x80x9cp53xe2x80x9d or xe2x80x9cp53 activityxe2x80x9d refers to p53 protein. The invention is believed to work by temporarily suppressing expression of the p53 gene and/or activity of p53 protein.